F12-23_DNA_ladder.jpgDNA Sequencing "Ladder" in an autoradiogram

    Besides the ddNTPs, the DNA sequencing reaction formerly incorporated a radioactively - labelled dNTP. The radioactive label exposes a sheet of X-ray film laid on top of the transferred gel as an autoradiogram. The DNA sequence is read from bottom to top, according to the known order of the four termination reactions at top.

    The sequences of both DNA strands are typically determined and compared as a check on accuracy. The sequence of the second strand will be the reverse complement of the first, which makes direct comparison tedious. Arrangement of the four reactions in the order shown (GTAC) allows the base sequence of the complementary strand on a second autoradiogram to be read directly in the same order as the first strand, by turning the autoradiogram over, and reading from top to bottom (think about it!).

    Homework: Read the sequence of this DNA ladder. Sequencing labs in Bio2250 was conducted like this, < 1991.

Figure 2000 by Griffiths et al. ; text 2013 by Steven M. Carr